The anthrax bacillus produces a three-component exotoxin of which an essential element for bioactivity is the protein known as protective antigen, or PA. PA binds to a cell surface receptor and is cleaved to generate a 63 kDa protein to which the one of the other anthrax toxins, LF and EF, can bind. Proteolysis and binding of PA permits internalization of a PA-LF complex into the cytosol, where it is able to attack cellular machinery, resulting in cell death. Since proteolytic cleavage of the PA anthrax toxin is obligatory for the manifestation of toxic activity, this cleavage step represents a natural target for pharmacologic intervention. Previous research has shown that this cleavage is performed by a cellular surface enzyme known as furin, a member of the family of eukaryotic subtilisins. This application is directed toward the idea that the cytotoxic action of anthrax toxin can be attenuated through inhibition of the activating cleavage event, resulting in lessened toxicity and cellular protection. In the last decade, several groups have shown that it is possible to block cleavage of bacterial toxins using engineered protein inhibitors of furin. We have recently used combinatorial chemistry techniques to identify a stable hexapeptide, D6R {D-hexa-arginine) which represents a potent, stable small molecule inhibitor of furin. Our preliminary data indicate that D6R can effectively inhibit furin-mediated cleavage of a bacterial toxin derived from Pseudomonas, blocking lethal effects in both cell lines as well as live animals. We here propose to systematically apply our studies of furin inhibition to the blockade of anthrax toxin activation. Specifically, we will investigate the use of D6R itself as a potential therapeutic in the attenuation of anthrax toxin cytotoxicity; examine the structural requirements for inhibition of furin-mediated cytotoxicity by D6R related molecules; and test D6R and/or other stable furin inhibitors identified in this work in animal models of anthrax toxicity. Our preliminary data showing potent inhibition of PA cytotoxicity by D6R support the idea that small molecule furin inhibitors will represent effective agents for the biologic attenuation of anthrax toxin cytotoxicity, the development of such antitoxin agents will add significantly to our ability to protect organisms against this pathogen.